Parkinson's disease (PD) is the second most common neurodegenerative disease and afflicts roughly one million people in the United States alone. This number is equivalent to 1-2% of the population over the age of 65. The neuropathological characterization of PD includes not only the loss of dopaminergic neurons in the substantia nigra and other brainstem nuclei but also the presence of cytoplasmic inclusions called Lewy bodies (LB) and abnormal alpha-synuclein-positive axonal swellings called Lewy neurites (LN) in surviving neurons. Little is known about how LB or LN are formed, how they affect neuron survival or what role they play in the pathogenesis of PD. Studies of autopsy tissue have provided little more than a catalog of LB and LN constituents. Other animal and cell models based on mutations in familial PD also have not produced inclusions that meet the criteria for LB found in PD brain. Our group has generated a model of sporadic PD by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA (mtDNA) to create cytoplasmic hybrid (cybrid) cell lines. In a longterm culture model, cybrid lines bearing PD mtDNA generate fibrillar and vesicular cybrid Lewy bodies (CLB) some of which are concentric (i.e. have a core and halo). CLB form spontaneously in PD cybrids and contain alpha-synuclein and replicate the essential antigenic and structural features of LB in PD brain. In preliminary studies, differentiated, neuronal PD cybrids also spontaneously form neuritic swellings that resemble LN, which have been named cybrid Lewy neurites (CLN). In Project 4 of this application proliferating PD and control (CNT) cybrid lines will be used to explore what role altered proteolysis (decreased proteasome activity, decreased lysosome activity, and increased expression of misfolded proteins or alpha-synuclein) contribute to the formation of CLB in PD cybrid cells. It will also be determined if CLB or CLN disrupt axonal transport of organelles, seek to determine if CLB are functional sites of protein degradation and if CLB expression can be reduced in PD cybrid cell lines. A clear understanding of how PD neuropathological changes like LB and LN are formed will ultimately help devise better treatment options.